Trinucleotide Repeat Instability via DNA Damage and Repair

描述（由申请人提供）：遗传性三核苷酸重复 (TNR) 不稳定性（即扩展和缺失/收缩）与 40 多种人类家族性神经退行性疾病和癌症相关。非遗传性体细胞 TNR 不稳定性可能与公众中这些疾病的发生有关。目前还没有针对 TNR 相关疾病的有效治疗方法，部分原因是对其潜在机制了解甚少。我们最近发现DNA碱基损伤和碱基切除修复（BER）通过诱导单链DNA（ssDNA）断裂和促进GC自碱基配对发夹的形成来启动和调节体细胞CAG重复扩增和缺失。这表明 DNA 碱基损伤、ssDNA 断裂和 BER 在调节 TNR 不稳定性中的新作用。为了探索 DNA 损伤和 BER 作为预防和治疗 TNR 相关疾病的新靶点的潜力，在本项目中，我们试图了解环境和化疗引起的 ssDNA 断裂及其低效修复如何与 BER 期间的体细胞 TNR 不稳定有关。这一目标将通过追求三个具体目标来实现。目标 1 是确定环境和化疗诱导的 DNA 碱基损伤和 ssDNA 断裂的积累是否会优先导致位点特异性的 CAG 重复不稳定。将确定由环境毒物和化疗剂（例如氯乙烯和替莫唑胺）诱导的 CAG/CTG 重复序列中 ssDNA 断裂的位点特异性积累。 DNA 损伤剂诱导的 ssDNA 断裂积累的独特模式将与重复扩增和缺失相关联，以识别损伤特异性对 CAG 重复不稳定性的“位置效应”。将在 BER 酶和辅因子水平不平衡的情况下进一步检查其影响，以确定是否可以通过降低 BER 效率来调节 TNR 不稳定性。目标 2 是检验以下假设：低效 BER 通过促进多个非 B 型 DNA 结构的形成而促进 CAG 重复删除。这将通过确定 DNA 聚合酶（Pol ß 遗传变体，Pol κ）的低效 DNA 合成是否会促进模板发夹的积累并促进 TNR 删除来完成。目标 3 是确定是否可以通过 BER 蛋白-蛋白相互作用和功能协调有效破坏非 B 型 DNA 结构来防止 TNR 扩增和缺失。该项目通过剖析环境和化疗 DNA 损伤、BER 和 TNR 不稳定性之间的相互作用，探讨 DNA 损伤引起的体细胞 TNR 不稳定性的基本机制。这些结果将为了解环境和化疗应激如何影响普通人群中 TNR 相关人类疾病的发生和进展，以及如何通过 DNA 损伤修复来预防这些不利影响提供重要的新见解。这将有助于确定TNR相关疾病预防、诊断和治疗的新靶点，并为环境和化疗引起的基因毒性效应的风险评估提供新信息。

DESCRIPTION (provided by applicant): Inherited trinucleotide repeat (TNR) instability, (i.e. expansions and deletions/contractions) is associated with more than 40 human familial neurodegenerative diseases and cancer. Non-inherited somatic TNR instability may be involved in the development of these diseases in the general public. No effective treatment for TNR- related diseases is yet available, partially because of a poor understanding of the underlying mechanisms. We have recently discovered that DNA base damage and base excision repair (BER) initiate and modulate somatic CAG repeat expansion and deletion by inducing single-strand DNA (ssDNA) breaks and promoting the formation of GC self-base-pairing hairpins. This indicates a new role of DNA base lesions, ssDNA breaks and BER in modulating TNR instability. To explore the potential of DNA damage and BER as new targets for the prevention and treatment of TNR-related diseases, in this project we seek to understand how environmentally and chemotherapeutically induced ssDNA breaks and their inefficient repair are involved in somatic TNR instability during BER. This goal will be achieved by pursuing three Specific Aims. Aim 1 is to determine if the accumulation of environmentally and chemotherapeutically induced DNA base lesions and ssDNA breaks can preferentially lead to CAG repeat instability in a site-specific manner. Site-specific accumulation of the ssDNA breaks in CAG/CTG repeat tracts induced by environmental toxicants and chemotherapeutic agents such as vinyl chloride and temozolomide will be determined. The unique patterns of ssDNA break accumulation induced by DNA-damaging agents will be correlated with repeat expansion and deletion to identify damage- specific "position effects" on CAG repeat instability. The effects will be further examined under imbalanced levels of BER enzymes and cofactors to determine if TNR instability can be modulated by compromised BER efficiency. Aim 2 is to test the hypothesis that inefficient BER facilitates CAG repeat deletion by promoting the formation of multiple non-B-form DNA structures. This will be done by determining if inefficient DNA synthesis by DNA polymerases (Pol ß genetic variants, Pol κ) can facilitate the accumulation of a template hairpin and promote TNR deletion. Aim 3 is to determine if TNR expansion and deletion can be prevented by efficiently disrupting non-B-form DNA structures through BER protein-protein interactions and functional coordination. This project addresses the fundamental mechanisms underlying DNA damage-induced somatic TNR instability by dissecting the interplay among environmental and chemotherapeutic DNA damage, BER, and TNR instability. The results will provide important new insights into how exposure to environmental and chemotherapeutic stresses may influence the development and progression of TNR-related human diseases in the general population, and how these adverse effects can be prevented by DNA damage repair. This will help to identify novel targets for prevention, diagnosis, and treatment of TNR-related diseases, and provide new information for risk assessment of environmentally and chemotherapeutically induced genotoxic effects.